Team:Heidelberg/Implementation
Proposed implementation
Our Tools in the Real World
Introduction
In the end, what is it all for? A key element of science has always been not only to pursue truth but to harness the knowledge gained from research and use it for the common good. We think that our project can be more than just an interesting experiment confined to a laboratory. We believe that the tools we developed can also be used in exciting applications in the real world. Here we want to propose ways in which they could help solving pressing problems or advance areas of research in synthetic biology.Virus mediated Drug Delivery
Every pharmacist must ask himself how his therapeutic drug is going to reach the location inside the patient's body where it is supposed to act. Currently, complex layers of packaging with different chemicals are required to send the active agent to its target, without it being degraded on the way or taking its effect at another location. This makes the process of drug delivery one further obstacle in fighting diseases. Luckily, there are biological constructs that are well equipped to target structures with high specificity and at the same time fight degradation – viruses. Much effort has been put into research focused on redesigning them to carry therapeutics or evolving them to target specific human tissue. With success! Today phage therapy or therapy with adenoviruses has proven to be useful against many health threats like antibiotic-resistant bacteria or cancer.
However there are still problems that arise with the usage of viruses as vehicles for modern therapeutical approaches like gene-therapy – their limited size. The frequently used Adeno Associated Viruses (AAV) vector is limited to 5 kbp which can be transferred and translated in the targeted cells Domenger2019NextgenerationAV . This is not enough room for large protein complexes with high therapeutic potential - like the recently published prime editors Anzalone2019SearchandreplaceGE . Different labs already proposed ways for creating synthetic capsids like the Dietz lab, which is currently developing methods for assembling stable transport vesicles from DNA origami DietzOrigami . Although the preliminary results are extremely promising, there are still issues concerning the possible biodegradation by the patient’s own immune system as well as efficient targeting using these structures. Another large obstacle origins in the current regulations on drug approval. Like any original medical application, the large synthetic scaffolds need to pass the guidelines of good development practice for medical products by the European Commission, FDA and other drug controlling organizations which aim to ensure the safety of the end patients. Since the new technology is built out of completely synthetic parts each element needs to go through rigorous testing and pre- and clinical trials to first ensure that it gives new harm. Next efficient production and purification of the scaffolds, as well as the encapsulation of the drug compound, need to be established on a pharmaceutical scale. These processes are necessary, but also take valuable time - sometimes more than 15 years morgan2011cost .
This is where our Protein RNA Interaction project could shine. Using small RBPs like BBa_K3113010 or BBa_K3657014 together with the expression of RNA Linkers there would be no need to fit a whole protein complex into one large vector. The protein complex could instead be split into functional subunits, distributed into two different AAV vectors, delivered separately, and then assembled post-translationally in the targeted cell Patel2019DesignOA . This would allow the pharmaceutical companies to use already well-established production technology for AAV vectors with just a few minor adjustments. This strategy of presenting a new complex drug in an old package offers a way to decrease R&D costs which would otherwise inevitably come with establishing a completely new delivery strategy based solely on synthetic compounds.
Still, it could be challenging to ensure the highest possible amount of association between the protein complex subunits to ensure the full effect. Of course, as always when developing new drugs the guidelines of good development practice for medical products by the European Commission, FDA and other drug controlling organization have to be followed to ensure the safety for patients that would be treated with the drug. When these guidelines would be followed properly, we would not expect safety dangers that could arise from this application.
Diversifying Cellular Circuit Design
Although complicated artificial biological circuits exist already, this field of synthetic biology is still extensively researched and longs for more biological parts that can extend its design capabilities. For example, much effort is put into designing independent small molecule sensors with minimum cross talk, to enable the precise activation and repression of targeted genes
With these parts, we provide a foundational advance that could initially only be of use in the laboratory but could influence many white and green biotechnological applications down the road in which biological circuits are utilised.
PRISM and 3DOC simplify the design of RNA-binding proteins and Ribonucleic-protein complexes.
Our tools PRISM (Protein RNA interaction sequence modeling), 3DOC (3D-domain concatenation) are designed for academic users. As part of our toolbox we address other scientists and provide them with tools for their future work.PRISM enables them to generate sequences of RNA-binding proteins, which bind a specific user-chosen RNA and fulfil characteristics, such as the charge or amount of certain amino acids. By this a specific design of a RNA-binding protein is possible. As stated above the design of Protein-RNA interaction sites is especially important in AAV vectors
3DOC is a pipeline to generate 3D-models of modular proteins, such as RNA-binding Pumby and PPR proteins, for instance. With our tool the Protein-RNA complex modelling is significantly simplified, so researchers can easily check for steric hinders. The output of PDBs can be used for example for Ribonucleic protein complex generation modelling via Rosetta
RISE is made for iGEMers and synthetic biologists.
The iGEM registry extends the tools of synthetic biologists by a full-stack catalogue of biobricks. Over the years motivated teams filled it up with multiple originally designed parts and complex structures often providing characterizations together with the sequences. But as it often happens with databases which are being used and edited by multiple people without generalized supervision, the database becomes more difficult to use. Multiple parts are present completely without characterization and annotation. The original categories that can be found on the wiki now do not completely represent the data that can be found on the separate pages. The search after keywords also became complex.
This is why our team created RISE (iGEM Registry intelligent search engine) as a new standard and easy-to-use tool for all iGEM teams and other users of the iGEM registry at universities and research institutes. RISE will enable scientists to filter and search for biobricks by a keyword or certain characteristics. RISE also offers the possibility to export the sequences as well as annotations in various file formats. This functionality significantly enhances the experience of the iGEM Registry.
Still, there is a lot of work to be done for future iGEM teams to make the registry a better place. Using our search engine you can now easily search for uncharacterized parts that can be linked to your project and successfully fulfil your medal criteria and provide data in a more targeted way to a larger amount of parts and thus fill the currently blank pages with valuable information featuring positive and negative results. Further, you can leave keywords and hashtags featuring experiments that still need to be done. This would help future teams to realize what experiments are missing for characterization and evolve their projects around them again enhancing the overall quality of the data in the registry.
To achieve all of that we hope to implement the tool directly into the iGEM webpage, so it can be downloaded and used by everyone interested.